Clearance of persistent SARS-CoV-2 associates with increased neutralizing antibodies in advanced HIV disease post-ART initiation

SARS-CoV-2 clearance requires adaptive immunity but the contribution of neutralizing antibodies and T cells in different immune states is unclear. Here we ask which adaptive immune responses associate with clearance of long-term SARS-CoV-2 infection in HIV-mediated immunosuppression after suppressive antiretroviral therapy (ART) initiation. We assembled a cohort of SARS-CoV-2 infected people in South Africa (n = 994) including participants with advanced HIV disease characterized by immunosuppression due to T cell depletion. Fifty-four percent of participants with advanced HIV disease had prolonged SARS-CoV-2 infection (>1 month). In the five vaccinated participants with advanced HIV disease tested, SARS-CoV-2 clearance associates with emergence of neutralizing antibodies but not SARS-CoV-2 specific CD8 T cells, while CD4 T cell responses were not determined due to low cell numbers. Further, complete HIV suppression is not required for clearance, although it is necessary for an effective vaccine response. Persistent SARS-CoV-2 infection led to SARS-CoV-2 evolution, including virus with extensive neutralization escape in a Delta variant infected participant. The results provide evidence that neutralizing antibodies are required for SARS-CoV-2 clearance in HIV-mediated immunosuppression recovery, and that suppressive ART is necessary to curtail evolution of co-infecting pathogens to reduce individual health consequences as well as public health risk linked with generation of escape mutants.

The notable findings, carried out using robust methodology are of clearance on appearance of neutralizing antibodies and binding antibodies that also correlated with detection of dolutegravir in patient samples.Also, the emergence of virus evolution and immune escape in chronic infection (mostly in previously well-described sites) is also a very important observation.The use of in vitro assays of patient samples and also hamsters to look at the impact of XBB 1.5 antibodies is very useful, alongside the inclusion of appropriate controls.
This study highlights the risk associated with untreated or poorly treated HIV infection in the generation of variants with a shift in antigenic profile and resultant changes in phenotype.It also highlights the impact of ARV (dolutegravir-based) therapy in reversing this risk.
The manuscript and figures are highly polished and clear.I have a few fairly minor questions/comments.1.How many people in the HIV+ COVID cohort became chronically infected with SARS-CoV-2 -are we in a position to comment on this risk in more detail?The systematic design of this study is really important and the associated risk has not previously been quantified, while case studies/series have previously been reported (although none quite as well as this).I note also from the protocol that transmission was considered.Were the highly evolved lineages transmitted?2. Minor point.In the x axis of Figure 1 timelines.are each of the intervals correctly labelled or have they slipped a bit?The 2021 timeline seems to start with a short period and then there another 4 -are these meant to be continuous timelines e.g.~2 months each?? 3. Given that HIV causes T cell deficiency, it would have been useful to see some data on ELISpot or flow cytometry responses as well as the neutralization data -this should be acknowledged as a limitation of the study.
Reviewer #2 (Remarks to the Author): This manuscript by Karim et al. describes the long-term SARS-CoV-2 infection in people living with HIV.HIV-mediated immunosuppression can alter the SARS-CoV-2 infection dynamics, and virus clearance can be delayed significantly.They have shown that in people with advanced HIV disease, SARS-CoV-2 infection can persist for 100-200 days.Interestingly, they found that suppressing HIV viremia using ART led to the clearance of SARS-CoV-2.This clearance was associated with the emergence of neutralizing antibodies.They also report that the SARS-CoV-2 vaccine was effective only when HIV has been suppressed.People with active viral replication or low CD4+ T cell counts failed to respond to the vaccine.This study highlights how immunosuppressed people, particularly those living with advanced HIV disease, can be persistently infected with SARS-CoV-2.Authors have shown that neutralizing antibodies are associated with protection against SARS-CoV-2, as shown by several other studies.Importantly, the level of HIV suppression and CD4+ count in HIVinfected people might offer a way to predict vaccine efficacy in these individuals.Overall, the authors used a unique cohort of patients and have done a longitudinal study to understand SARS-CoV-2 infection dynamics.The findings reported here will be helpful to further understand the interplay between HIV and SARS-CoV-2 infection.As long-term COVID is now becoming a public health problem, this study will offer some insight to further understand SARS-COV-2 persistence and clearance in immunosuppressed individuals.
I believe the manuscript can be further improved, and there are some key issues that need to be addressed.
Major points: 1)The clearance of SARS-CoV-2 is associated with an increase in FRNT titer, a decrease in HIV genome copies, and an increase in CD4 count.The authors have highlighted that clearance was associated with an increase in neutralizing activity.However, it might be possible that CD4+ cell increase may lead to increased CD8+ activity.Is it possible to look at CD8+ T cell activity in these individuals?
2)It is not clear why neutralizing antibodies appear after 100-200 days of infection.One explanation is that an increase in CD4+ T cells might have improved B cell affinity maturation, leading to potent neutralizing antibody production.However, those individuals might still be responding to infection and generating non-neutralizing antibodies even when they have low levels of CD4+ T cells.It will be important to look at different antibody isotypes (IgM, IgG, and IgA) to see if those individuals were making antibodies to SARS-CoV-2.
3)The clearance of SARS-CoV-2 and HIV RNA coincides with the initiation of ART.Is there any direct effect of ART on SARS-CoV-2?Has it been shown that ART does not inhibit SARS-CoV-2 replication?If not, an in vitro test can be performed to show the effect of these drugs on SARS-CoV-2.
Minor points 1)Line 50 -the sentence seems incomplete 2)Line 66-67 -the sentence needs to be rephrased 3)Show correlation plots to show the correlation between SARS-CoV-2 genome copies, neutralizing antibodies, CD4+ T cell count, and HIV genome copies.

4)Fig 2. X-axis label missing
5)How many times FRNT were performed?6)Can you include the conclusion for every result section?The way it is written now makes it very hard to follow the paper.If you include a conclusion for every section, it would be easier for the readers to transition from one section to another.
Reviewer #3 (Remarks to the Author): The study follows 5 participants who live with advanced HIV disease and are immunosuppressed over their courses of SARS-COV-2 infection.Data are presented for their HIV infection, viremia control, CD4 cells counts, and their SARS-COV-2 infection and clearance, vaccination, and binding and neutralizing antibody responses.The study provide strong evidence the association between neutralizing antibody response development and SARS-COV-2 virus clearance in these participants, and also demonstrated the need for HIV viremia control and sufficient CD4 T cell presence for the development of function neutralizing response against SARS-COV-2.Data are clearly presented.Conclusions drawn are well-supported.Findings of substantial evolution of SARS-COV-2 virus in immunosuppressed subjects over long infection period is consistent with other researchers finding.The strong association between neutralizing response and SARS-COV-2 virus clearance is important for the field.The observations that a "normal" vaccine responses can be elicited in people living with advanced HIV disease provided that viremia is controlled and CD4 T cell population is reconstituted should be informational for clinical practices involving advance HIV infections.

Questions:
1.The association between timing of neutralizing antibody response and virus clearance if clear in the study.However, timing of neutralizing antibody response also coincide with CD4 T cell reconstitution in most cases.Have SARS-COV-2 specific T cell responses being studied in these participants?HIV viremia control can be associated with both better CD4 health and CD8 T cell response.It would be great if we can see whether T cell response is also involved in the SARS-COV-2 clearance.I do understand such investigation can be limited by specimen availability though.2. Neutralizing antibody response development is associated with CD4 T cell reconstitution in most cases.Therefore CD4 T cell count also strongly associated with virus clearance in most cases.However in Pt 255, CD4 T cells returned to normal range at D237, whereas neutralizing response and virus clearance didn't happen until D293.It may worth noting in discussion that at least in this case, CD4 T cell reconstitution itself isn't sufficient for virus clearance.Minor comments: 3. Line 193 "Participant 255 cleared SARS-CoV-2 at the vaccine baseline visit…" and then Line 195 "Participant 255 still had SARS-CoV-2 infection at vaccination".This is very confusing.Is the Participant 255 on line 195 supposed to be Participant 209? 4. Line 198 "but not at two weeks post-vaccination, the first post-vaccination timepoint tested and expected peak….".Is this referring to only after the first vaccination?It looks like neutralizing response did increase at the first time point tested after the 2nd vaccination.5. Line 211 "and this was also observed for participant 209".This refers to "strong increase in antispike antibodies but not neutralization".However for Pt 209, there is not really a strong increase in anti-spike antibodies following vaccination.The peak binding response that shows an increase is at Day 0 of 1st vaccination, which is most likely from infection.The binding response did not increase until much later on.
       7,16,42,48,49 .One cause of immunosuppression which has been shown in case studies to lead to SARS-CoV-2 long-term infection and evolution is uncontrolled HIV infection resulting in extensive CD4 T cell depletion, termed advanced HIV disease 4-6,15  3)Show correlation plots to show the correlation between SARS-CoV-2 genome copies, neutralizing antibodies, CD4+ T cell count, and HIV genome copies.
There are not enough autologous virus neutralization data points to make an informative correlation plot unless we combine participants, and this would mask the heterogeneity of participant responses.We therefore prefer to keep the SARS-CoV-2 titer/neutralizing antibody association figure the way it is.               FRNT 50        6)Can you include the conclusion for every result section?The way it is written now makes it very hard to follow the paper.If you include a conclusion for every section, it would be easier for the readers to transition from one section to another.
We thank the Reviewer for the suggestion.We now include the following summaries at the end of each section of the Results: Section: Advanced HIV disease leads to long-term SARS-CoV-2 infection and evolution              significantly prolonged SARS-CoV-2 infection.Secondly, during the time of prolonged SARS-CoV-2 infection, there is an accumulation of mutations, some of which are known to lead to     Section: Clearance of prolonged SARS-CoV-2 infection associates with emergence of neutralization Summary         -CoV-2 clearance and emergence of a neutralizing response.Complete HIV suppression was not required, and neutralization was likely mediated by IgG but not IgA isotypes, with the limitation that the              Section: No association with SARS-CoV-2 specific CD8 T cell responses           -CoV-2 specific CD8 T cell responses are involved in SARS-CoV-2 clearance during recovery from advanced HIV   Section: Poor vaccine elicited neutralization in advanced HIV disease participants with HIV viremia            neutralization response in participants without advanced HIV disease as well as those with advanced HIV disease but who already controlled their HIV infection, it did not perform well in eliciting SARS-CoV-2 neutralization in the two advanced HIV disease participants with HIV  Section: Hamster infection shows evolved virus from Delta variant infection is antigenically distinct           -CoV-2 infection in advanced HIV disease immunosuppression can lead to the evolution of SARS-CoV-2 which has extensive antigenic differences relative to both past and currently circula  We thank the Reviewer for picking this up.The neutralizing antibody response did increase for BA.1 but not the other viruses.This is corrected on lines 303-307 in the current version:               except Omicron BA.1 at two weeks post-second dose.BA.1 neutralization did increase slightly to FRNT50=82) two weeks post-vaccination which is the expected peak of the                          anti-               increase in anti-spike antibodies following vaccination.The peak binding response that shows an increase is at Day 0 of 1st vaccination, which is most likely from infection.The binding response did not increase until much later on.

3 :
Section: Virus evolved from Delta escapes Delta but not Omicron XBB-elicited neutralization               in this study was antigenically distinct from the Omicron XBB.1.5subvariant, this virus did not escape neutralization resulting from XBB-derived subvariant infections or relatively recent  Reviewer #Data are clearly presented.Conclusions drawn are well-supported.Findings of substantial evolution of SARS-COV-2 virus in immunosuppressed subjects over long infection period is consistent with other researchers finding.The strong association between neutralizing response and SARS-COV-2 virus clearance is important for the field.The observations that a             disease provided that viremia is controlled and CD4 T cell population is reconstituted should be informational for clinical practices involving advance HIV infections.cases.However in Pt 255, CD4 T cells returned to normal range at D237, whereas neutralizing                           ent for virus clearance.This is an important point, and we now add these details to lines 245-247 of the Results:               showed CD4 T cell reconstitution which plateaued at day 237 post-diagnosis, a time when SARS-CoV-2 was not yet cleared.Clearance was detected on day 293 (Figure 2D, second   We add this point to the Discussion on lines 405-411:             reconstitution, as measured by CD4 T cell concentrations in the blood, in 4 out of 5 advanced HIV disease participants.However, in participant 255, CD4 T cell reconstitution peaked at day 237 post-SARS-CoV-2 diagnosis, whereas the neutralizing response and virus clearance happened on day 293.At least for this case, CD4 T cell reconstitution was not sufficient for virus clearance, or clearance was delayed relative to CD4 recons Minor comments:       -CoV-              -CoV-       confusing.Is the Participant 255 on line 195 supposed to be Participant 209?We thank the Reviewer for picking this up.Corrected on lines 299-300 in the current version:               255 cleared SARS-CoV-2 at the vaccine baseline visit, before vaccination was administered (Figure 1B).Participant 209 had SARS-CoV-2 infection at vaccination (second dose, Figure          -vaccination, the first post-vaccination timepoint tested                neutralizing response did increase at the first time point tested after the 2nd vaccination.